Non-naturally occurring synthetic lytic peptides

ABSTRACT

Non-naturally occurring lytic peptides which contain a phenylalanine residue and one or more alanine, valine and lysine residues, and optionally contain chemically masked cysteine or serine residues possess an amphipathic structure which allows them to promote cell lysis in certain pathologic organisms, and particularly in prokaryotes. Peptides having a beta-pleated sheet secondary structure and lacking cysteine residues form one embodiment of these lytic peptides.

This application is a division of Ser. No. 08/427,001, filed Apr. 21,1995, now U.S. Pat. No. 5,717,064, which is a continuation-in-part ofSer. No. 08/148,889, filed Nov. 8, 1993 (abandoned), and acontinuation-in-part of Ser. No. 08/689,489, filed Aug. 12, 1996, nowU.S. Pat. No. 6,001,805, which is a continuation of 08/231,730, filedApr. 20, 1994, now U.S. Pat. No. 5,561,107, which is acontinuation-in-part of Ser. No. 08/225,476, filed Apr. 8, 1994(abandoned), which is a continuation-in-part of 08/039,620, filed Jun.4, 1993 (abandoned), and Ser. No. 08/148,889, filed Nov. 8, 1993(abandoned), which is a continuation-in-part of Ser. No. 08/148,491,filed Nov. 8, 1993 (abandoned).

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to methylation-stabilized, lysine-richsynthetic lytic peptide compositions with enhanced resistance toproteolytic digestion, and to methods of making the same.

DESCRIPTION OF RELATED ART

Naturally occurring lytic peptides play an important if not criticalrole as immunological agents in insects and have some, albeit secondary,defense functions in a range of other animals. The function of thesepeptides is to destroy prokaryotic and other non-host cells bydisrupting the cell membrane and promoting cell lysis. Common featuresof these naturally occurring lytic peptides include an overall basiccharge, a small size (23-39 amino acid residues), and the ability toform amphipathic α-helices. Several types of naturally occurring lyticpeptides have been identified: cecropins (described in U.S. Pat. Nos.4,355,104 and 4,520,016 to Hultmark et al.), defensins, sarcotoxins,melittin, and magainins (described in U.S. Pat. No. 4,810,777 toZasloff). Each of these peptide types is distinguished by sequence andsecondary structure characteristics.

Several hypotheses have been suggested for the mechanism of action ofthe lytic peptides: disruption of the membrane lipid bilayer by theamphipathic α-helix portion of the lytic peptide; lytic peptideformation of ion channels, which results in osmotically inducedcytolysis; lytic peptide promotion of protein aggregation, which resultsin ion channel formation; and lytic peptide-induced release ofphospholipids. Whatever the mechanism of lytic peptide-induced membranedamage, an ordered secondary conformation such as an amphipathic α-helixand positive charge density are features that appear to participate inthe function of the lytic peptides.

Active analogs of naturally occurring lytic peptides have been producedand tested in vitro against a variety of prokaryotic and eukaryotic celltypes (see for example Arrowood, M. J., et al., J. Protozool. 38: 161s[1991]; Jaynes, J. M., et al., FASEB J. 2: 2878 [1988]), including: grampositive and gram negative bacteria, fungi, yeast, envelope viruses,virus-infected eukaryotic cells, and neoplastic or transformed mammaliancells. The results from these studies indicate that many of thesynthetic lytic peptide analogs have similar or higher levels of lyticactivity for many different types of cells, compared to the naturallyoccurring forms. In addition, the peptide concentration required to lysemicrobial pathogens such as protozoans, yeast, and bacteria does notlyse normal mammalian cells.

The specificity of the lytic action depends upon the sequence andstructure of the peptide, the concentration of the peptide, and the typeof membrane with which it interacts. Jaynes J. M. et al., PeptideResearch 2: 157 (1989) discuss the altered cytoskeletal characteristicsof transformed or neoplastic mammalian cells that make them susceptibleto lysis by the peptides. In these experiments, normal, humannon-transformed cells remained unaffected at a given peptideconcentration while transformed cells were lysed; however, when normalcells were treated with the cytoskeletal inhibitors cytochalasin D orcolchicine, sensitivity to lysis increased. The experiments show thatthe action of lytic peptides on normal mammalian cells is limited. Thisresistance to lysis was most probably due to the well-developedcytoskeletal network of normal cells. In contrast, transformed celllines which have well-known cytoskeletal deficiencies were sensitive tolysis. Because of differences in cellular sensitivity to lysis, lyticpeptide concentration can be manipulated to effect lysis of one celltype but not another at the same locus.

Synthetic lytic peptide analogs can also act as agents of eukaryoticcell proliferation. Peptides that promote lysis of transformed cellswill, at lower concentrations, promote cell proliferation in some celltypes. This stimulatory activity is thought to depend on thechannel-forming capability of the peptides, which somehow stimulatesnutrient uptake, calcium influx or metabolite release, therebystimulating cell proliferation (see Jaynes, J. M. Drug News &Perspectives 3: 69 [1990]; and Reed, W. A. et al., MolecularReproduction and Development 31: 106 [1992]). Thus, at a givenconcentration, these peptides stimulate or create channels that can bebeneficial to the normal mammalian cell in a benign environment where itis not important to exclude toxic compounds.

The synthetic lytic peptide analogs according to the present inventiontypically contain as few as 12 and as many as 40 amino acid residues. Aphenylalanine residue is often positioned at the amino terminus of theprotein to provide an aromatic moiety analogous to the tryptophanresidue located near the amino terminus of natural cecropins and aUV-absorbing moiety with which to monitor the purification of thesynthetic peptide. The basis for the design of these lytic peptideanalogs is that a peptide of minimal length, having an amphipathicα-helical structural motif, and overall positive charge density effectslytic activity. Peptides that have the structural motif of a β-pleatedsheet and overall positive charge density can also effect lyticactivity.

As discussed above, in vitro laboratory tests of the lytic peptideanalogs have been successful. However, the use of the lytic peptideanalogs in vivo could be considerably limited in circumstances whereproteases may digest the peptide analogs before sufficient pathogen celllysis has occurred. In particular, the high concentration of positivelycharged amino acids such as lysine and arginine make the syntheticpeptides susceptible to tryptic digestion. The secondary conformation ofthe peptides sequesters the hydrophobic amino acid residues, thusshielding them from interaction with proteases such as chymotrypsin,which hydrolyzes peptides at bulky or aromatic amino acid residues. Thisproteolytic susceptibility is a general problem for peptides andproteins when used in vivo. Many techniques are suitable for stabilizingproteins to tryptic digestion for in vitro use but are not appropriatefor in vivo or oral administration to humans and animals.

Several studies teach that modification by methylation of the N-terminalα-amino group in a protein or peptide has been used to studystructure-function relationships in a variety of naturally occurringproteins and their substrates or receptors.

Means, G. E. et al., Biochemistry 7: 2192 (1968) teach that whenproteins are treated with aldehydes or ketones and sodium borohydride,amino groups are converted into corresponding mono- or dialkylaminoderivatives. Trypsin attacks proteins and peptides at the positivelycharged lysine and arginine residues. Dimethylation of the ε-amino groupof lysine residues renders some modified proteins essentially resistantto tryptic digestion, however, in this study, the test enzymeribonuclease was enzymatically inactivated by the modification.

Lilova, A. et al., Biol. Chem. Hoppe-Seyler 368: 1479 (1987) teach thatmethylation of ε-amino groups of lysine residues can be used todetermine the essential nature of lysine residues in the maintenance ofbiological activity. This report states that the lysine residues in thetest protein (subtilisin DY) do not participate directly in thecatalytic reaction.

Boarder et al., Biochem. Pharmacol. 30: 1289 (1981) teach that theN-terminal α-amino group and the ε-amino group of lysine residues inβ-endorphin, a naturally occurring opioid peptide, can be dimethylatedusing formaldehyde and sodium cyanoborohydride, providing resistance totryptic and aminopeptidase digestion. The authors speculate that thenaturally occurring, modified β-endorphin may have a longer half-life invivo. As a basis for this speculation the authors cite Hammond et al.,1979, in which the authors report that for β-endorphin, receptor bindingis maintained after lysine ε-amino dimethylation. However, thisstatement of retained receptor affinity cannot be extrapolated topredict bioreactivity of the methylated derivative. The biologicalactivity of the modified β-endorphin was tested in neither case.

Coy et al., U.S. Pat. No. 5,059,653 teach a solid state method usingsodium cyanoborohydride and carbonyl-containing compounds to modify theε-amino group of lysine residues to provide proteolytic stability forproteins. However, the use of sodium cyanoborohydride introduces intothe preparation a potential cyanide contamination that may bedetrimental for in vivo usage. Furthermore, this method does not addressretention of biological activity.

Accordingly, it would be a significant advance in the art to provide amethod of producing methylated physiologically active lytic peptidesthat have enhanced resistance to proteolysis.

It would be particularly desirable to provide a method of producing suchpeptides so that the methylated peptides have enhanced proteolyticstability and retain their physiological activity for in vivoapplications against pathogenic microbial organisms such as bacteria,yeast, fungi, and protozoans; neoplastic or transformed cells; envelopeviruses; and virally-infected cells.

These and other objects and advantages will be more fully apparent fromthe ensuing disclosure and claims.

SUMMARY OF THE INVENTION

The present invention relates generally to methylation-stabilized,lysine-rich synthetic lytic peptide compositions with enhancedresistance to proteolytic digestion, and to methods of making the same.

More specifically, the present invention relates in a broadcompositional aspect to chemically modified, lysine-rich syntheticpeptides with an overall positive charge density, wherein the ε-aminogroups of lysine residues and the N-terminal α-amino group aredimethylated by a method of reductive alkylation.

In one particular aspect, the present invention relates to aphysiologically active peptide composition comprising a syntheticphysiologically active peptide that has been chemically modified,wherein the ε-amino groups of lysine residues and the N-terminal α-aminogroup are sufficiently methylated such that the chemically modifiedphysiologically active peptide has enhanced in vivo resistance toenzymatic digestion, relative to the physiologically active peptidealone.

In another aspect, the present invention relates to a group of similarphysiologically active peptide compositions, comprising: (i)physiologically active synthetic peptides that have been chemicallymodified, wherein the ε-amino groups of lysine residues and theN-terminal α-amino group are dimethylated; and (ii) chemically modified,physiologically active synthetic peptides that are related by amino acidsequence and physiological activity.

In another aspect, the invention relates to a physiologically activepeptide composition comprising synthetic peptides which contain as fewas 12 and as many as 40 amino acid residues, wherein the peptide iscomprised mainly of three amino acid monomers: alanine, valine andlysine. A phenylalanine residue is present at or near the N-terminus ofthe peptide. Cysteine or serine amino acid residues are present in fiveof the peptides and have chemically masked side chain groups.

The invention relates in a further aspect to a physiologically activepeptide composition comprising a physiologically active syntheticpeptide that has been chemically modified, wherein the ε-amino groups oflysine residues and the N-terminal α-amino group are dimethylated. Insuch a peptide, the secondary conformation of the peptide is in aperiodic structural motif, an amphipathic α-helix, in which one side ofthe cylinder is hydrophilic, with the polar amino acid residues exposedon this surface. The other side of the cylinder is hydrophobic, with theside chains of the hydrophobic amino acid residues seeking an anhydrousenvironment. Serine residues may be exposed on either surface.

The invention relates in a further aspect to a physiologically activepeptide composition comprising a physiologically active syntheticpeptide that has been chemically modified, wherein the ε-amino groups oflysine residues and the N-terminal α-amino group are dimethylated. Insuch a peptide, the secondary conformation of the peptide is in aperiodic structural motif, the β-pleated sheet, in which the polypeptidechain is extended in a sheet-like conformation rather than acylinder-like conformation.

The invention relates in yet a further aspect to a physiologicallyactive peptide composition comprising a physiologically active syntheticpeptide that has been chemically modified, wherein the ε-amino groups oflysine residues and the N-terminal α-amino group are dimethylated. Insuch a peptide, the secondary conformation is an amphipathic α-helix.With such a conformation in an aqueous environment, the hydrophobicregions would adhere to each other to form micelles and hence isolateddomains of a separate phase. Only the dimethylated lysine side chainresidues that are hydrophilic but have enhanced resistance to tryptichydrolysis are exposed to the aqueous environment, and hence toproteolytic enzymes.

The invention relates in yet a further aspect to a physiologicallyactive peptide composition comprising a physiologically active syntheticpeptide that has been chemically modified, wherein the ε-amino groups oflysine residues and the N-terminal α-amino group are dimethylated. Insuch a peptide, the secondary conformation of the peptide is in aperiodic structural motif, the β-pleated sheet. In such a configuration,individual polypeptides can associate into overlapping structures. Thisassociation is stabilized by hydrogen bond formation between NH and COgroups in separate polypeptide strands.

The invention relates in a further aspect to a physiologically activepeptide composition comprising a physiologically active syntheticpeptide that has been chemically modified, wherein the ε-amino groups oflysine residues and the N-terminal α-amino group are dimethylated. Sucha peptide is used in vivo to treat infections caused by pathogenicmicrobial organisms such as bacteria, yeast, fungi, and protozoans bylysing these organisms; to treat cancers caused by neoplastic ortransformed cells by lysing such cells; and to treat viral infections bylysing envelope viruses and virally-infected cells.

The term “amphipathic” as used herein refers to the distribution ofhydrophobic and hydrophilic amino acid residues along opposing faces ofan α-helix structure or other secondary conformation, which results inone face of the α-helix structure being predominantly hydrophobic andthe other face being predominantly hydrophilic. The degree of amphipathyof a peptide can be assessed by plotting the sequential amino acidresidues on an Edmunson helical wheel.

The term “peptide” as used herein is intended to be broadly construed asinclusive of polypeptides per se having molecular weights of up to10,000 daltons, as well as proteins having molecular weights of greaterthat about 10,000 daltons, wherein the molecular weights are numberaverage molecular weights.

The term “methylated” as used herein means that the specified aminogroups have been chemically reacted by a method of reductive alkylationor methylation so that the associated hydrogen atoms are replaced bycovalently coupled methyl groups.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the TNBS assay standard curve for unmodified peptideShiva-11.

FIG. 2 shows the modified-TNBS assay standard curve for unmodifiedpeptide DP-1.

FIG. 3 shows the modified-TNBS assay spectrophotometric data before andafter trypsin digestion of unmodified and modified DP-1 peptide.

FIG. 4 shows net values of the data from FIG. 3A after subtraction ofthe trypsin autodigestion control sample from the appropriateexperimental samples.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

Chemical modification of lytic peptide analogs offers certainadvantages. If the modifications are made in such a way that the lyticpeptides retain all or most of their biological activity, then thefollowing advantage results: the peptides have enhanced stability toproteolysis. With enhanced stability, the peptides can be administeredin vivo without loss of biological activity through proteolyticdigestion.

The chemical modification of lysine rich lytic peptide analogs bymethylation of the ε-amino groups of lysine residues and the N-terminalα-amino group achieves in vivo stabilization against tryptic attackwhile preserving biological activity.

When considering lytic peptide analog stabilization with chemicalmodification of amino acid residue side chains, it is important toconsider the character (hydrophobic or hydrophilic) and location of theindividual amino acids residues within the peptides of concern. With thelytic peptide analogs proposed herein, the following are the only typesof amino acid residues to be examined: phenyalanine, alanine, valine,lysine, cysteine, and serine. Of this group, serine and lysine arepotentially exposed to proteases in the aqueous environment, as a resultof the secondary conformation of the peptide. In peptides containingserine or cysteine, these groups could be previously chemically modifiedto mask amino acid residue.

The lytic peptide analogs are designed to take the configuration of anamphipathic α-helix structure or, in the case of SEQ ID 30-38, aβ-pleated sheet conformation. In an aqueous environment the hydrophobicregions of these peptides would adhere to each other, forming micellesand hence isolated domains of a separate phase. In this circumstance,the hydrophobic moieties would be unavailable to the aqueous phase andhence to hydrolysis by proteolytic enzymes. In one preferred aspect ofthe invention, the lytic peptides assume the secondary conformation ofan amphipathic α-helix.

Each lysine side chain contains an ε-amino group (—NH₃ ⁺) which providesthe peptide with a unit positive charge at physiological pH. Thecombined charges from the multiple lysine side chains contribute to thepolarity and thus the regional hydrophilicity required for formation ofan amphipathic α-helix. The positive charge of these lytic peptideanalogs is required for activity. Amphipathy alone does not provide forlytic action, thus the SEQ ID 30-38 peptides in the β-pleated sheetconformation with an overall positive charge density also have lyticactivity. Modification of the ε-amino group of the lysine amino acidresidue does not affect the unit charge of the lysine residue or thepeptide. However, susceptibility to tryptic hydrolysis for the lysineresidue α-carbonyl peptide linkages is drastically reduced.

As discussed above, it can be presumed that alanine, valine, internalphenylalanine, cysteine, and serine contained in the lytic peptideanalogs are not vulnerable to proteolytic attack due to their removalfrom the aqueous phase or prior chemical masking. Lysine, however,provides a specific locus for the most aggressive proteolytic enzyme,trypsin. For this reason, reductive alkylation of the lytic peptideswould provide enhanced stability to proteolytic hydrolysis. It shouldalso be noted that the N-terminal α-amino group is also exposed andwould also become alkylated during such a procedure, thus providingfurther resistance to both chymotrypsin, which attacks aromatic aminoacids such as phenylalanine, and aminopeptidases, which act at theN-terminus.

One objective of the present invention is to provide enhancedproteolytic stability to a series of lysine-rich, lytic peptide analogs.Another objective is to use such modified lytic peptides for in vivodelivery of physiologically effective lytic peptides.

The features and advantages of the invention are more fully shown by thefollowing illustrative examples and embodiments, which are not to belimitingly construed as regards the broad scope, utility, andapplicability of the invention.

EXAMPLE 1 Representative Lytic Peptides

Set out in Table 1 below as illustrative examples of lytic peptideanalogs of the present invention are the amino acid sequences of afamily of related peptide analogs. The peptides may be synthesizedaccording to conventional methods using a Milligen™ solid phase peptidesynthesizer. Representative peptides from this group are methylated, andused in subsequent experimental examples. The three letter amino acidsymbols are as follows: Ala, alanine; Cys, cysteine; Lys, lysine; Phe,phenylalanine; Ser, serine; and Val, valine. These lytic peptide analogsare designated for ease of reference as SEQ ID NO. 1-6 and 14-43.

Table 1: Peptide Sequences

SEQ ID NO. 1: Phe Ala Val Ala Val Lys Ala Val Lys Lys Ala Val Lys LysVal Lys Lys Ala Val Lys Lys Ala Val Lys Lys Lys Lys

SEQ ID NO. 2: Phe Ala Val Ala Val Lys Ala Val Ala Val Lys Ala Val LysLys Ala Val Lys Lys Val Lys Lys Ala Val Lys Lys Ala Val Lys Lys Lys Lys

SEQ ID NO. 3: Phe Ala Val Ala Val Lys Ala Val Ala Val Lys Ala Val AlaVal Lys Ala Val Lys Lys Ala Val Lys Lys Val Lys Lys Ala Val Lys Lys AlaVal Lys Lys Lys Lys

SEQ ID NO. 4: Phe Ala Val Ala Val Lys Ala Val Lys Lys Ala Val Lys LysVal Lys Lys Ala Val Lys Lys Ala Val

SEQ ID NO. 5: Phe Ala Val Ala Val Lys Ala Val Ala Val Lys Ala Val LysLys Ala Val Lys Lys Val Lys Lys Ala Val Lys Lys Ala Val

SEQ ID NO. 6: Phe Ala Val Ala Val Lys Ala Val Ala Val Lys Ala Val AlaVal Lys Ala Val Lys Lys Ala Val Lys Lys Val Lys Lys Ala Val Lys Lys AlaVal

SEQ. ID NO. 14: Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Leu Ala ArgLys Iso Ala Arg Leu Gly Val Ala Phe

SEQ ID NO. 15: Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Lys Val LysLys Val Ala Lys Lys Val Ala Lys Val Ala Val Ala Val

SEQ ID NO. 16: Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Lys Val LysLys Val Ala Lys Lys Val Ala Lys Val Ala Val Ala Lys Val Ala Val Ala Val

SEQ ID NO. 17: Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Lys Val LysLys Val Ala Lys Lys Val Ala Lys Val Ala Val Ala Lys Val Ala Val Ala LysVal Ala Val Ala Val

SEQ ID NO. 18: Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val Ala LysLys Val Ala Lys Val Ala Val Ala Val

SEQ ID NO. 19: Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val Ala LysLys Val Ala Lys Val Ala Val Ala Lys Val Ala Val Ala Val

SEQ ID NO. 20: Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val Ala LysLys Val Ala Lys Val Ala Val Ala Lys Val Ala Val Ala Lys Val Ala Val AlaVal

SEQ ID NO. 21: Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Val Ala LysLys Val Ala Lys Val Ala Lys Lys Val Ala Lys Lys Val

SEQ ID NO. 22: Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Val Ala LysLys Val Ala Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala

SEQ ID NO. 23: Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Val Ala LysLys Val Ala Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala Lys Lys Val AlaLys Val Ala Lys Lys

SEQ ID NO. 24: Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val Ala LysVal Ala Lys Lys Val Ala Lys Lys Val

SEQ ID NO. 25: Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val Ala LysVal Ala Lys Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala

SEQ ID NO. 26: Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val Ala LysVal Ala Lys Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala Lys Val Ala LysLys

SEQ ID NO. 27: Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val Ala LysVal Ala Lys Lys Val Ala Lys Lys Val Lys Lys Lys Lys

SEQ ID NO. 28: Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val Ala LysVal Ala Lys Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala Lys Lys Lys Lys

SEQ ID NO. 29: Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val Ala LysVal Ala Lys Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala Lys Val Ala LysLys Lys Lys Lys Lys

SEQ ID NO. 30: Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys Lys LysLys Lys

SEQ ID NO. 31: Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys Ala LysVal Lys Ala Lys Lys Lys Lys

SEQ ID NO. 32: Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys Ala LysVal Lys Ala Lys Val Lys Ala Lys Val Lys Lys Lys Lys

SEQ ID NO. 33: Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys

SEQ ID NO. 34: Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys Ala LysVal Lys Ala

SEQ ID NO. 35: Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys Ala LysVal Lys Ala Lys Val Lys Ala Lys Val

SEQ ID NO. 36: Lys Lys Lys Lys Phe Lys Val Lys Ala Lys Val Lys Ala LysVal Lys

SEQ ID NO. 37: Lys Lys Lys Lys Phe Lys Val Lys Ala Lys Val Lys Ala LysVal Lys Ala Lys Val Lys Ala

SEQ ID NO. 38: Lys Lys Lys Lys Phe Lys Val Lys Ala Lys Val Lys Ala LysVal Lys Ala Lys Val Lys Ala Lys Val Lys Ala Lys Val

SEQ ID NO. 39: Phe Lys Lys Val Lys Lys Val Ala Lys Lys Val Cys Lys CysVal Lys Lys Ala Val Lys Lys Val Lys Lys Phe

SEQ ID NO. 40: Phe Ala Val Ala Val Lys Ala Val Lys Lys Ala Val Lys LysVal Lys Lys Ala Val Lys Lys Ala Val Cys Cys Cys Cys

SEQ ID NO. 41: Cys Cys Cys Cys Phe Val Lys Lys Val Ala Lys Lys Val LysLys Val Ala Lys Lys Val Ala Lys Val Ala Val Ala Val

SEQ ID NO. 42: Phe Ala Val Ala Val Lys Ala Val Lys Lys Ala Val Lys LysVal Lys Lys Ala Val Lys Lys Ala Val Ser Ser Ser Ser

SEQ ID NO. 43: Ser Ser Ser Ser Phe Val Lys Lys Val Ala Lys Lys Val LysLys Val Ala Lys Lys Val Ala Lys Val Ala Val Ala Val

Chemical modification of lytic peptide analogs offers certainadvantages. If the modifications are made in such a way that thepeptides retain all or most of their lytic characteristics, then thephysiologically active peptides have enhanced stability to proteolysis.With enhanced stability, oral delivery of the peptide is advantageouslyaccommodated without excessive loss of activity due to proteolyticdigestion.

EXAMPLE 2 Chemical Modification by Methylation

An exemplary and preferred reaction scheme for reductive alkylation oflysine residue ε-amino group and the N-terminal α-amino group isdescribed below.

The preferred method for reductive alkylation uses pyridine borane asthe reducing agent. This reagent is one of a class of reducing agentsknown as amine boranes. Pyridine borane exhibits a slightly higherreducing capacity than sodium cyanoborohydride, another reducing agentthat can be used for the reductive alkylation. Pyridine borane drivesthe reductive alkylation reaction to complete dimethylation with nomonomethyl products when excess reagents are used, as demonstrated byWong, W. S. D., et al. Analytical Biochemistry 139: 58 (1984). While asmuch as 25% of cyanoborohydride goes to N-cyanomethyl products, loweringits methylation yield, pyridine borane does not appear to be involved inany such secondary reaction. In addition, sodium cyanoborohydrideprovides the potential hazard of contaminating the product with cyanide,severely limiting its use in therapeutic and in vivo applications. Thealkylation reagent may suitably comprise formaldehyde as a methyl group(methylation) precursor. Shown below are the agents of reductivealkylation, formaldehyde and pyridine borane, the substrate, peptidyllysine, and the chemical formulae of the reaction scheme species.

Reaction Scheme for Dimethylation of Peptidyl Lysine

A representative lysine containing peptide, DP-1 (SEQ ID No. 44) (amellitin analog), was used as the test substrate for the reductivealkylation reaction. DP-1 is a 23-mer lytic peptide with the sequencePhe-Ala-Leu-Ala-Leu-Lys-Ala-Leu-Lys-Lys-Ala-Leu-Lys-Lys-Leu-Lys-Lys-Ala-Leu-Lys-Lys-Ala-Leu.The peptide (20 mg) was dissolved in 1.6 ml 0.2 M HEPES buffer(N-2-hydroxyehylpiperazine-N′-2-ethane sulfonic acid), pH 7.0. While themixture was stirring, 0.2 ml of 1.2 M pyridine borane (0.750concentrated pyridine borane in 5 ml HPLC grade methanol) was added.Next, 0.2 ml of 0.726 M formaldehyde (0.6 ml 37% formaldehyde [HCHO] in10 ml HEPES pH 7.0 buffer) was added to the mixture. A trace(approximately 1 μl) of 1-octanol was included in the reaction volume tocontrol foaming. The reaction volume was then stirred for 2 hours atroom temperature. After 2 hours the reaction mixture was titrated tobelow pH 3.0 with 0.2 M HCl. The reaction mixture was then frozen andlyophilized to reduce volume, and the resulting residue was washed 3times with anhydrous ether to remove the pyridine borane. The reactionresidue was reconstituted to approximately 2.0 ml with 0.1 M acetic acidand applied to a 2.4 cm×31 cm G-15-120μ Sephadex™ column to purify thereaction product. After the calibrated front eluted from the column (0.1M acetic acid was the elution reagent), 20 ml of eluate containing theproduct was collected and the eluate was lyophilized to dryness.

The peptide was stored at −20° C. in the presence of a desiccant astheir acetate salt. For use in the following examples, modified peptidesare dissolved in a saline buffer, pH 7.0, at a concentration of 0.1mg/ml to 10 mg/ml.

EXAMPLE 3 Determination of Lysyl Dimethylation

The method selected to monitor the level of reductive alkylation is aprocedure which provides a spectrophotometric assay of the unsubstitutedamino groups (Habeeb, A. F. S. A., Anal. Biochem., 14: 328 [1966]). Thisprocedure uses 2,4,6-trinitrobenzenesulfonic acid (TNBS, picrylsulfonicacid) which reacts with primary amines to form trinitrophenyl (TNP)derivatives, producing a visible yellow color and a UV absorbancemaximum at 335 nm. The extent of amino group substitution can thereforebe quantified. The procedure is described as follows: Added to the assaytubes is 1.0 ml of 4% NaHCO₃, pH 8.5. With mixing, 1.0 ml of peptidestandards and unknowns at a concentration of 0.06 to 1.0 mg/ml peptideare added to separate tubes and then 1.0 ml of 0.1% TNBS is added toeach tube. The solutions are incubated at 40° C. for 45-60 minutes.After the incubation period, 0.5 ml of 1N HCL is added to the tubes. Ifnecessary, 1 ml of 10% sodium dodecyl sulfate may be added prior to theaddition of the acid to prevent precipitation of the peptides. Theabsorbance at 335 nm is read versus a blank of the reagent mixture minuspeptide. As shown in the figure below, the standard curve for detectionof unmodified amino groups is essentially linear up to 1.0 mg/ml. Theunmodified peptide used for the standard curve is Shiva-11 (SEQ. ID NO:45), a cecropin analog. The sequence of this 31-mer peptide is:Phe-Ala-Lys-Lys-Leu-Ala-Lys-Lys-Leu-Lys-Lys-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Leu-Ala-Leu-Ala-Leu-Lys-Ala-Leu-Ala-Leu-Lys-Ala-Leu.The TNBS assay standard curve is shown in FIG. 1.

When 0.15 mg of a representative peptide (DP-1), which had beenalkylated using the pyridine borane procedure described above, wassubjected to the TNBS assay for unmodified amino groups, it yielded anabsorbance of 0.007 (compare to standard curve in above figure). Thisabsorbance is essentially zero, meaning that the amino groups on thepeptide are fully methylated. In comparison, a reference unmodifiedpeptide sample (Shiva-11) showed an absorbance of 0.474 under the sameconditions, indicating a 100% free amino groups. Additionally, thealkylated peptide and its unmodified counterpart were subjected toanalytical reversed phase column chromatography and compared with theunmodified peptide. Both scans showed a single peak with no satellitepeaks or shoulders.

EXAMPLE 4 In Vitro Lysis of Pathogenic Bacteria

The effect of lytic peptides (SEQ ID NO. 14 and DP-1) were testedagainst pathogenic bacteria in vitro. In this test, antibiotic resistantclinical isolates of Pseudomonas aeruginosa and Staphylococcus aureuswere obtained. The lytic peptide bioassay was performed as describedbelow.

A flask containing 49 ml of nutrient broth was inoculated with 1 ml ofan overnight culture of the test bacteria. The culture was allowed togrow to mid-log phase at 37° C. with shaking (approximately 4 hours).When the cells reached the correct density, the cells were transferredto a sterile tube and centrifuged for 10 minutes at 3000 rpm. The pelletwas resuspended in 3 ml of phosphate buffer and centrifuged for 10minutes at 3000 rpm. The pellet was resuspended once again in sufficient(but measured) volume to calculate the absorbance of the suspension at600 nm. Using the resulting absorbance and a previously constructedgrowth curve, the required dilution to achieve a concentration of 10⁶cells/ml was determined.

One micromole of the test peptide was dissolved in 1.0 ml of 0.01%acetic acid to make a 1 mM solution and serial dilutions were made togive a range of peptide concentrations from 10 μM to 1 mM. The testculture tubes for the bioassay contained 800 μl of phosphate buffer, pH7.0, 100 μl of cells at 10⁶ cells/ml and 100 μl of peptide solution (10μM to 1 mM). The final concentration of peptide in the assay was from 1μM to 100 μM. A reaction system minus peptide was included as a control.The tubes were incubated at 37° C. for one hour.

After the incubation period two 1:10 serial dilutions in phosphatebuffer were made for each culture(three 1:10 serial dilutions for thecontrol culture). 1.00 μl of each dilution was spread on a nutrient agarplate, in duplicate and incubated overnight at 37° C. The following day,the number of colonies on the control plates was counted to determinethe starting number of cells in the assay tubes. The number of cellssurviving the assay in the presence of peptide was also counted. Theresults are shown in Table 2.

TABLE 2 BACTERICIDAL ACTIVITY OF MODIFIED AND UNMODIFIED PEPTIDES WITH ACLINICAL ISOLATE OF PSEUDOMONAS AERUGINOSA Peptide Control μMModification^(a) # of Survivors % of minus peptide  0 100,000 100 SEQ IDNO. 14 10 2 0.002 SEQ ID NO. 14 10 m, g 91 0.09 SEQ ID NO. 14  1 100,000100 SEQ ID NO. 14  1 m, g 100,000 100 DP-1 10 6,607 6.6 DP-1 10 m 2,0422 DP-1  1 100,000 100 DP-1  1 m 100,000 100 ^(a)m = methylated lysineresidues, g = glyoxylated arginine residues.

This data in this table show that the modification of the peptides doesnot affect their bacteriolytic activity. Each peptide has a differentextent of bacteriolytic activity for a given bacterial species at agiven concentration. In general, the peptides in this experimentdemonstrated bacteriolytic activity at a concentration of 10 μM, but notat a concentration of 1 μM.

TABLE 3 BACTERICIDAL ACTIVITY OF MODIFIED AND UNMODIFIED PEPTIDES WITH ACLINICAL ISOLATE OF STAPHYLOCOCCUS AUREUS Peptide Control μMModification^(a) # of Survivors % of minus peptide  0 50,000 100 SEQ IDNO. 14 10 2,299 4.6 SEQ ID NO. 14 10 m, g 2,818 5.6 SEQ ID NO. 14  150,000 100 SEQ ID NO. 14  1 m, g 50,000 100 DP-1 10 813 1.6 DP-1 10 m891 1.8 DP-1  1 50,000 100 DP-1 1 m 50,000 100 ^(a)m = methylated lysineresidues, g = glyoxylated arginine residues.

This data in this table show that the modification of the peptides doesnot affect their bacteriolytic activity. Each peptide has a differentextent of bacteriolytic activity for a given bacterial species at agiven concentration. In general, the peptides in this experimentdemonstrated activity at a concentration of 10 μM, but not at aconcentration of 1 μM.

EXAMPLE 5 Peptide Minimal Inhibitory Concentration

Nutrient broth was inoculated with bacteria and incubated for severalhours at 37° C. The density of the incubated broth was then adjusted toa MacFarland 0.5 turbidity standard by adding test broth. Thestandardized suspension was diluted 1:100. A stock solution of each testantimicrobial agent (DP-1 and methylated DP-1) was prepared in distilledwater at a concentration of 1 mg/ml. This stock solution was thendiluted with test broth to yield solutions with concentrations of 32μg/ml, 16 μg/ml, 8 μg/ml, 4 μg/ml, and 2 μg/ml each in 0.9 ml ofnutrient broth. To each drug solution 0.1 ml of inoculum was added, andone growth control tube prepared (0.9 ml nutrient broth with 0.1 mlinoculum). For purity check a loopful of the growth control tube wasstreaked for isolation to a SBAP. All tubes were examined for visibleturbidity of growth and the Minimal Inhibitory Concentration (MIC) wasdetermined. The MIC of the tested drug is defined as the lowestconcentration of the drug which inhibits the growth of the organism.

TABLE 4 MIC OF A LYTIC PEPTIDE FOR VARIOUS PATHOGENIC BACTERIA ClinicalIsolate MIC DP-1 MIC Methylated DP-1 1. Staphylococcus aureus A 16 >16 B 8 >16 C  8 >16 D 16 >16 E 16 >16 2. Enterococcus species  1  4  16  2 8  16  3  4  8  4  4  16  5 16  16 3. Pseudomonas aeruginosa  2  2  215  8 >16 30  4  8 31  4  8 32  4  16 4. Xanthomonas sp.  4  4  2  5 16 16  6  4  4  8 >16   16

The data in this table show that although the MIC of the methylated andunmethylated peptides vary slightly in some cases, for the most part thedifferences between the MIC values for the methylated and unmethylatedpeptides are negligible.

EXAMPLE 6 Fungicidal Activity of Modified and Unmodified Peptides

The following is a description of the formation of alginate beads forfungicide testing. Fungal spores/conidia were suspended in approximately10 ml of a 20% sterile malt extract solution. The suspension was thenfiltered through a double layer of cheese cloth (or similar material) inorder to remove mycelial fragments. 10 ml of a 2% sodium alginatesolution was then added to 10 ml of the fungal spore suspension.Approximately 50 ml of a 10% malt extract solution which contains 1%calcium chloride was poured into a 100 ml beaker. The fungalspore/alginate solution was then added slowly to the calcium chloridesolution using a syringe fitted with a fine needle which allows thesolution to be added in a drop-wise manner. It is important that thechloride solution is continuously stirred so that the alginate beads donot stick together as they mature. In solution the calcium ions replacethe sodium ions, which leads to the formation of the jelly-like beads.The beads are left in the chloride solution for at least 30 minutesbefore use to ensure that the ion exchange has been completed. The beadsformed by this method have a size of about 3 mm in diameter (dependenton the size of the needle used). The beads contain the fungal spores andthe necessary nutrients for development. For the test of peptidefungicide activity, two beads are added to 200 μl of the peptidesolution in a 96 well microtiter plate. The peptide concentration rangesfrom 1 mM to 5 μM (1 mM, 0.5 mM, 0.1 mM, 0.05 mM etc.). The assay isvisually scored for mycelium growth on Day 2. The result of this assaywith unmodified DP-1 and methylated DP-1m are shown in Table 5 below.

TABLE 5 FUNGICIDAL ACTIVITY OF A LYTIC PEPTIDE Peptide Concentration μMFungal Species Growth minus peptide Pyrolaria + DP-1   5 Pyrolaria +DP-1  10 Pyrolaria + DP-1  50 Pyrolaria − DP-1m   5 Pyrolaria + DP-1m 10 Pyrolaria +/− DP-1m  50 Pyrolaria − minus peptide Botrytis + DP-1 100 Botrytis + DP-1  500 Botrytis +/− DP-1 1000 Botrytis − DP-1m  100Botrytis + DP-1m  500 Botrytis + DP-1m 1000 Botrytis +

The data in the table above show that modification of the peptide bymethylation does not have a significant effect on its fungicidalactivity, although the peptides themselves have variable activitydepending on the fungal species. Both unmodified and methylated DP-1 hasfungicidal activity for Pyrolaria at a concentration of 50 μM, and theactivity would likely been demonstrated between 10 and 50 μM. Incontrast, even at high concentrations, both unmodified and methylatedDP-1 lack fungicidal activity for Botrytis.

EXAMPLE 7 Proteolytic Resistance of Modified Peptides to TrypsinDigestion

A. Modification of the TNBS Assay

In Example 3, a TNBS assay was used to compare unmodified Shiva-11 andmodified DP-1 to demonstrate the success of the alkylation reaction.Although this method is adequate for detection of α-amino groups in mostpeptides, there is at least one application for which it is not. Thelimitation of the assay was exposed in an attempt to determine thesusceptibility of the unmodified peptide, DP-1, to tryptic digestion. InExample 3, unmodified DP-1 was not tested with the TNBS assay.

After incubation at 40° C., the TNBS-treated DP-1 peptide, which had notbeen subjected to trypsin hydrolysis, produced an opalescence whichgradually formed a flocculent precipitate, obscuring the truespectrophotometric value of the sample. According to Habeeb, A. S. F.A., Anal. Biochem. 14: 328 (1966), this problem can be remedied byadding sodium dodecyl sulfate to the assay cuvet. In this instance,however, the prescribed remedy was not effective. Alteration of the pHof the assay above and below the recommended 8.5 was also not effectivein eliminating this problem. Therefore, to complete the experiments forExample 7, it was necessary to revise the TNBS assay to accommodate theanomalous behavior of the unmodified DP-1 peptide.

DP-1 and its methylated form, DP-1m, were both soluble in pyridine.Furthermore, the reaction of TNBS with primary amines to form thetrinitrophenyl (TNP) derivatives could be conducted in pyridine alonewithout the addition of NaHCO₃, pH 8.5, as specified in the originalprocedure of Example 3. To prevent any opalescence formation, only asmall amount of H₂O could be introduced in this assay. Due to the UVabsorptive quality of pyridine, absorption for this modified assay wasmeasured at 440 nm in the visible region (the chromophore produces abright yellow color), rather than the near UV range (335 nm) asspecified in the original assay procedure. The TNP derivatizationrequired a much shorter incubation period to reach maximum absorption inthe modified assay procedure.

The modified assay procedure is as follows. Add 2.5 ml purified pyridine(with contaminating aniline removed) into a cuvet. Add a maximum of 0.3ml of the aqueous peptide solution (0.02 to 0.25 mg). With vigorousmixing, add 0.01 ml of 1% TNBS solution in 1:1 pyridine:H₂O. Incubatefor 20 minutes at 40° C. Read the absorption at 440 nm and compare to ablank reaction minus peptide. FIG. 2 shows the standard curve forunmodified DP-1 using the modified TNBS assay.

B. Trypsin Digestion and Quantification with the Modified TNBS Assay

In this example, both DP-1 and its alkylated form DP-1m are exposed totryptic digestion, and then the amount of digestion is measured bydetermining the increase in the number of primary amine groups, usingthe modified TNBS assay. If alkylation of the peptide confers enhancedtryptic digestion resistance, then the modified DP-1m should show nofree amino groups in the TNBS reaction, while the unmodified DP-1 shouldshow an increase in the amount of free primary amine upon digestion withtrypsin. DP-1, the peptide used in this study, contains 23 amino acidresidues, of which 9 are lysine residues. The peptide contains noarginine. Thus, an unmodified peptide provides a total of 10 primaryamine groups before tryptic digestion.

Ten samples (1-10) were prepared. Trypsin was obtained from SigmaChemical Co. The activity is 9700 units/mg solid. The digestion buffercontains 0.01 M HEPES, pH 7.78, 0.05 M CaCl₂, and 0.10 M KCl. Thecomposition of the samples 1-10, respectively is shown in the tablebelow.

TABLE 6 TRYPSIN DIGESTION SAMPLES 1 2 3 4 5 6 7 8 9 10 Hepes Buffer 0.200.20 0.20 0.20 0.10 0.10 0.10 0.10 0.20 0.20 DP-1M 3 mg/ml 0.10 0.100.10 0.10 DP-1 3 mg/ml 0.10 0.10 0.10 0.10 Trypsin 1 mg/ml 0.10 0.100.10 0.10 0.10 0.10

The samples were incubated 30 minutes, after which 0.1 ml aliquots wereremoved for the modified TNBS assay as described in part A of thisexample. The TNBS assay shows the amount of free amino groups before andafter trypsin digestion. FIGS. 3 and 4 show the results of the trypsindigestion experiment.

The results of the TNBS assay after trypsin digestion demonstrate theexpected results. The alkylated DP-1 shows no change in the number ofprimary amino groups before and after digestion, demonstrating enhancedtrypsin digestion resistance. In contrast, the unmodified peptide showsan increase in the number of primary amino groups after trypticdigestion.

While the invention has been described herein, with certain features,and embodiments it will be recognized that the invention may be widelyvaried, and that numerous other modifications, variations, and otherembodiments are possible, and such modification, variations, and otherembodiments are to be regarded as being within the spirit and scope ofthe invention.

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 45(2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 27 (B) TYPE: AMINO ACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE:PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Phe Ala Val Ala Val LysAla Val Lys Lys Ala Val Lys Lys Val Lys 1 5 10 15 Lys Ala Val Lys LysAla Val Lys Lys Lys Lys 20 25 (2) INFORMATION FOR SEQ ID NO: 2: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 (B) TYPE: AMINO ACID (C)TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 2: Phe Ala Val Ala Val Lys Ala Val Ala Val Lys Ala Val LysLys Ala 1 5 10 15 Val Lys Lys Val Lys Lys Ala Val Lys Lys Ala Val LysLys Lys Lys 20 25 30 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 37 (B) TYPE: AMINO ACID (C) TOPOLOGY:LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Phe Ala Val Ala Val Lys Ala Val Ala Val Lys Ala Val Ala Val Lys 1 510 15 Ala Val Lys Lys Ala Val Lys Lys Val Lys Lys Ala Val Lys Lys Ala 2025 30 Val Lys Lys Lys Lys 35 (2) INFORMATION FOR SEQ ID NO: 4: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 (B) TYPE: AMINO ACID (C)TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 4: Phe Ala Val Ala Val Lys Ala Val Lys Lys Ala Val Lys LysVal Lys 1 5 10 15 Lys Ala Val Lys Lys Ala Val 20 (2) INFORMATION FOR SEQID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 (B) TYPE: AMINOACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 5: Phe Ala Val Ala Val Lys Ala Val Ala Val LysAla Val Lys Lys Ala 1 5 10 15 Val Lys Lys Val Lys Lys Ala Val Lys LysAla Val 20 25 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 33 (B) TYPE: AMINO ACID (C) TOPOLOGY:LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Phe Ala Val Ala Val Lys Ala Val Ala Val Lys Ala Val Ala Val Lys 1 510 15 Ala Val Lys Lys Ala Val Lys Lys Val Lys Lys Ala Val Lys Lys Ala 2025 30 Val (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCECHARACTERISTICS: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: This sequenceis intentionally skipped. (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCECHARACTERISTICS: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: This sequenceis intentionally skipped. (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCECHARACTERISTICS: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: This sequenceis intentionally skipped. (2) INFORMATION FOR SEQ ID NO: 10: (i)SEQUENCE CHARACTERISTICS: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Thissequence is intentionally skipped. (2) INFORMATION FOR SEQ ID NO: 11:(i) SEQUENCE CHARACTERISTICS: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:This sequence is intentionally skipped. (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: This sequence is intentionally skipped. (2) INFORMATION FOR SEQ IDNO: 13: (i) SEQUENCE CHARACTERISTICS: (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 13: This sequence is intentionally skipped. (2) INFORMATION FOR SEQID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 (B) TYPE: AMINOACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 14: Ala Val Lys Arg Val Gly Arg Arg Leu Lys LysLeu Ala Arg Lys Ile 1 5 10 15 Ala Arg Leu Gly Val Ala Phe 20 (2)INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:27 (B) TYPE: AMINO ACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Lys Lys Lys Lys Phe Val LysLys Val Ala Lys Lys Val Lys Lys Val 1 5 10 15 Ala Lys Lys Val Ala LysVal Ala Val Ala Val 20 25 (2) INFORMATION FOR SEQ ID NO: 16: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 (B) TYPE: AMINO ACID (C)TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 16: Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Lys Val LysLys Val 1 5 10 15 Ala Lys Lys Val Ala Lys Val Ala Val Ala Lys Val AlaVal Ala Val 20 25 30 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 37 (B) TYPE: AMINO ACID (C) TOPOLOGY:LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val 1 510 15 Ala Lys Lys Val Ala Lys Val Ala Val Ala Lys Val Ala Val Ala Lys 2025 30 Val Ala Val Ala Val 35 (2) INFORMATION FOR SEQ ID NO: 18: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 (B) TYPE: AMINO ACID (C)TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 18: Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val Ala LysLys Val 1 5 10 15 Ala Lys Val Ala Val Ala Val 20 (2) INFORMATION FOR SEQID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 (B) TYPE: AMINOACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 19: Phe Val Lys Lys Val Ala Lys Lys Val Lys LysVal Ala Lys Lys Val 1 5 10 15 Ala Lys Val Ala Val Ala Lys Val Ala ValAla Val 20 25 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 33 (B) TYPE: AMINO ACID (C) TOPOLOGY:LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val Ala Lys Lys Val 1 510 15 Ala Lys Val Ala Val Ala Lys Val Ala Val Ala Lys Val Ala Val Ala 2025 30 Val (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 27 (B) TYPE: AMINO ACID (C) TOPOLOGY:LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Lys Lys Lys Lys Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val 1 510 15 Ala Lys Val Ala Lys Lys Val Ala Lys Lys Val 20 25 (2) INFORMATIONFOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 (B)TYPE: AMINO ACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 22: Lys Lys Lys Lys Phe Val Lys Lys ValAla Lys Val Ala Lys Lys Val 1 5 10 15 Ala Lys Val Ala Lys Lys Val AlaLys Lys Val Ala Lys Lys Val Ala 20 25 30 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 (B) TYPE: AMINO ACID(C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 23: Lys Lys Lys Lys Phe Val Lys Lys Val Ala LysVal Ala Lys Lys Val 1 5 10 15 Ala Lys Val Ala Lys Lys Val Ala Lys LysVal Ala Lys Lys Val Ala 20 25 30 Lys Val Ala Lys Lys 35 (2) INFORMATIONFOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 (B)TYPE: AMINO ACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 24: Phe Val Lys Lys Val Ala Lys Val AlaLys Lys Val Ala Lys Val Ala 1 5 10 15 Lys Lys Val Ala Lys Lys Val 20 (2)INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:28 (B) TYPE: AMINO ACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: Phe Val Lys Lys Val Ala LysVal Ala Lys Lys Val Ala Lys Val Ala 1 5 10 15 Lys Lys Val Ala Lys LysVal Ala Lys Lys Val Ala 20 25 (2) INFORMATION FOR SEQ ID NO: 26: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 (B) TYPE: AMINO ACID (C)TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 26: Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val Ala LysVal Ala 1 5 10 15 Lys Lys Val Ala Lys Lys Val Ala Lys Lys Val Ala LysVal Ala Lys 20 25 30 Lys (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 27 (B) TYPE: AMINO ACID (C) TOPOLOGY:LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Phe Val Lys Lys Val Ala Lys Val Ala Lys Lys Val Ala Lys Val Ala 1 510 15 Lys Lys Val Ala Lys Lys Val Lys Lys Lys Lys 20 25 (2) INFORMATIONFOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 (B)TYPE: AMINO ACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 28: Phe Val Lys Lys Val Ala Lys Val AlaLys Lys Val Ala Lys Val Ala 1 5 10 15 Lys Lys Val Ala Lys Lys Val AlaLys Lys Val Ala Lys Lys Lys Lys 20 25 30 (2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 (B) TYPE: AMINO ACID(C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 29: Phe Val Lys Lys Val Ala Lys Val Ala Lys LysVal Ala Lys Val Ala 1 5 10 15 Lys Lys Val Ala Lys Lys Val Ala Lys LysVal Ala Lys Val Ala Lys 20 25 30 Lys Lys Lys Lys Lys 35 (2) INFORMATIONFOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 (B)TYPE: AMINO ACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 30: Phe Lys Val Lys Ala Lys Val Lys AlaLys Val Lys Lys Lys Lys Lys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 31:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 (B) TYPE: AMINO ACID (C)TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 31: Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys Ala LysVal Lys 1 5 10 15 Ala Lys Lys Lys Lys 20 (2) INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 (B) TYPE: AMINO ACID(C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 32: Phe Lys Val Lys Ala Lys Val Lys Ala Lys ValLys Ala Lys Val Lys 1 5 10 15 Ala Lys Val Lys Ala Lys Val Lys Lys LysLys 20 25 (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 12 (B) TYPE: AMINO ACID (C) TOPOLOGY:LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Phe Lys Val Lys Ala Lys Val Lys Ala Lys Val Lys 1 5 10 (2)INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:17 (B) TYPE: AMINO ACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: Phe Lys Val Lys Ala Lys ValLys Ala Lys Val Lys Ala Lys Val Lys 1 5 10 15 Ala (2) INFORMATION FORSEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 (B) TYPE:AMINO ACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 35: Phe Lys Val Lys Ala Lys Val Lys AlaLys Val Lys Ala Lys Val Lys 1 5 10 15 Ala Lys Val Lys Ala Lys Val 20 (2)INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:16 (B) TYPE: AMINO ACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: Lys Lys Lys Lys Phe Lys ValLys Ala Lys Val Lys Ala Lys Val Lys 1 5 10 15 (2) INFORMATION FOR SEQ IDNO: 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 (B) TYPE: AMINOACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 37: Lys Lys Lys Lys Phe Lys Val Lys Ala Lys ValLys Ala Lys Val Lys 1 5 10 15 Ala Lys Val Lys Ala 20 (2) INFORMATION FORSEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 (B) TYPE:AMINO ACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 38: Lys Lys Lys Lys Phe Lys Val Lys AlaLys Val Lys Ala Lys Val Lys 1 5 10 15 Ala Lys Val Lys Ala Lys Val LysAla Lys Val 20 25 (2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 25 (B) TYPE: AMINO ACID (C) TOPOLOGY:LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Phe Lys Lys Val Lys Lys Val Ala Lys Lys Val Cys Lys Cys Val Lys 1 510 15 Lys Ala Val Lys Lys Val Lys Lys Phe 20 25 (2) INFORMATION FOR SEQID NO: 40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 (B) TYPE: AMINOACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 40: Phe Ala Val Ala Val Lys Ala Val Lys Lys AlaVal Lys Lys Val Lys 1 5 10 15 Lys Ala Val Lys Lys Ala Val Cys Cys CysCys 20 25 (2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 27 (B) TYPE: AMINO ACID (C) TOPOLOGY:LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ IDNO:41: Cys Cys Cys Cys Phe Val Lys Lys Val Ala Lys Lys Val Lys Lys Val 15 10 15 Ala Lys Lys Val Ala Lys Val Ala Val Ala Val 20 25 (2)INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:27 (B) TYPE: AMINO ACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: Phe Ala Val Ala Val Lys AlaVal Lys Lys Ala Val Lys Lys Val Lys 1 5 10 15 Lys Ala Val Lys Lys AlaVal Ser Ser Ser Ser 20 25 (2) INFORMATION FOR SEQ ID NO: 43: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 (B) TYPE: AMINO ACID (C)TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 43: Ser Ser Ser Ser Phe Val Lys Lys Val Ala Lys Lys Val LysLys Val 1 5 10 15 Ala Lys Lys Val Ala Lys Val Ala Val Ala Val 20 25 (2)INFORMATION FOR SEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:23 (B) TYPE: AMINO ACID (C) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PEPTIDE(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: Phe Ala Leu Ala Leu Lys AlaLeu Lys Lys Ala Leu lys Lys Leu Lys 1 5 10 15 Lys Ala Leu Lys Lys AlaLeu 20 (2) INFORMATION FOR SEQ ID NO: 45: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 31 (B) TYPE: AMINO ACID (C) TOPOLOGY: LINEAR (ii) MOLECULETYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45: Phe Ala Lys LysLeu Ala Lys Lys Lys Lys Lys Leu Ala Lys Lys Leu 1 5 10 15 Ala Lys LeuAla Leu Ala Leu Lys Ala Leu Ala Leu Lys Ala Leu 20 25 30

What is claimed is:
 1. A non-naturally occurring lytic peptidecomprising a sequence of 12 to 40 amino acid residues which contains aphenylalanine residue, and one or more alanine, valine, and lysineresidues, and optionally contains chemically-masked cysteine or serineresidues, said lytic peptides selected from the group consisting ofSequence ID Nos. 1-6 and 14-38.
 2. A non-naturally occurring lyticpeptide wherein the lytic peptide has a beta-pleated sheet secondarystructure and wherein the lytic peptide lacks cysteine residues.
 3. Alytic peptide of claim 2 selected from the group consisting of SequenceID Nos. 30-38.